Insects are among humankind's most serious competitors for food and fiber resources. Approximately one third of worldwide agricultural production is lost to insect damage each year. Insects such as termites and carpenter ants cause millions of dollars in structural damage every year. Many serious human and animal diseases, including malaria, yellow fever, sleeping sickness, viral encephalitis, and plague, are transmitted by insects. Efforts to control insect pests have resulted in the development of a global insecticide industry with annual sales of approximately $6 billion. Most of these products are synthetic chemical neurotoxins such as chlorinated hydrocarbons (e.g., DDT), carbamates (e.g., carbaryl), organophosphates (e.g., malathion), and synthetic pyrethroids (e.g., cypermethrin). Relatively minor, though significant, chemical insecticides include insect growth regulators (e.g., diflubenzuron and methoprene) and metabolic disrupters (e.g., hydroxymethylnon).
Synthetic chemical insecticides are effective for controlling pest insects in a wide variety of agricultural, urban, and public health situations. Unfortunately there are significant, often severe, side effects associated with the use of these products. Many pest populations have developed significant resistance to virtually all chemical insecticides, requiring higher and higher rates of usage for continued control. In a number of severe cases, highly resistant pest populations have developed which cannot be controlled by any available product. Chemical insecticides may also have deleterious effects on non-target organisms. Populations of beneficial arthropods, such as predators and parasites, are sometimes more severely affected by chemical applications than the pests themselves. Minor pests, ordinarily held in check by these beneficial organisms, may become serious pests when their natural constraints are removed by the use of chemical insecticides. Thus, new pest problems may be created by attempts to solve established problems.
Chemical insecticides may also have adverse effects on vertebrates. The use of DDT has been banned in the United States, due primarily to the insecticide's great environmental persistence and its resulting tendency to accumulate in the tissues of predatory birds, thereby disrupting their ability to produce viable eggs. The use of carbofuran has been severely restricted due to its avian toxicity, and many species of fish are known to be quite sensitive to a variety of insecticides. A number of insecticides, such as methyl parathion, are also quite toxic to humans and other mammals, and by accident or misuse have caused a number of human poisonings. Clearly, the field of insect control would benefit greatly from the discovery of insecticides with improved selectivity for insects and reduced effects on non-target organisms.
The problems described above, along with other concerns including the possibility that some insecticides may act as human carcinogens, have created a strong demand for the development of safer methods of insect control. The practice of integrated pest management (IPM), which seeks to minimize the adverse environmental effects of chemical insecticides by relying on cultural and biological methods, is one response to this demand. The success of IPM, however, has been less than hoped due to the lack of effective biological alternatives to chemical insecticides. Because these alternatives can reduce the frequency and severity of pest outbreaks and delay the development of insecticide-resistant pest populations, their availability is critical to the success of IPM programs.
Insect pathogens have been the objects of much study as potential pest control agents. Generally, these pathogens are quite selective for insects and in many cases affect only a few closely related species of insects. A number of insect pathogens have been developed as products, including bacteria (e.g., Bacillus thuringiensis and Bacillus popiliae), viruses (e.g., nuclear polyhedrosis viruses) and protozoa (e.g., the microsporidian Nosema locustae). These products occupy only a small fraction of the insecticide market, however, due largely to their relatively slow action. Although pathogens may ultimately cause a high level of mortality in pest populations, the insects may take weeks to die and continue to feed for much of that time. Thus, an unacceptably high level of crop or commodity damage may be inflicted before control is achieved. Currently, researchers are actively seeking ways to improve the effectiveness of insect pathogens and other biological control tools.
Insecticidal toxins from arthropods have been the objects of increasing interest over the past decade. These materials have proved useful for the detailed study of neural and neuromuscular physiology in insects. They have also been used to enhance the effectiveness of certain insect pathogens. The insecticidal toxin AaIT, from the scorpion Androctonus australis, has been employed for both purposes. This toxin belongs to a group of peptides that are lethal to a variety of insects but have no detectable effect in mammals, even though they come from a species known to be dangerous to humans. Other toxins in A. australis venom are lethal to mammals but have no effect on insects. This selectivity is particularly interesting in view of the fact that both groups of toxins act on voltage-sensitive sodium channels. Understanding the molecular basis of this selectivity may lead to the development of chemical insecticides with reduced effects on mammals and other non-target organisms.
The effectiveness of insect pathogens has also been enhanced by the use of genes encoding AaIT and other insect-selective toxins. A number of reports have demonstrated that the insecticidal properties of the Autographa californica nuclear polyhedrosis virus (AcMNPV), a member of the baculovirus family, can be enhanced by modifying the viral genome to include a gene encoding an insecticidal toxin. Toxins employed for this purpose include AaIT, TxP-1 from the parasitic mite Pyemotes tritici, DTX9.2 from the spider Diguetia canities, and NPS-326 (now known as TaITX-1) from the spider Tegenaria agrestis. These toxins were inserted into the AcMNPV genome under the control of either the plo promoter or the polyhedrin promoter. Both promoters regulate the high-level expression of very late viral genes encoding component proteins of the viral occlusion bodies. In every case, recombinant viruses containing a toxin gene were more effective than the wild type virus, as measured by the time required for infected insects to die or become moribund.
Because the baculovirus system is well known to be a highly efficient and flexible method of expressing biologically active proteins from many different sources, it is reasonable to expect that newly discovered toxins will also be useful for enhancing the insecticidal activity of these viruses.
The use of these toxins is not expected to be limited to baculoviruses, however. Many other microbes, including bacteria and fungi, are known to be susceptible to such genetic manipulation. Certain bacteria and fungi, in fact, are widely used for large-scale production of exogenous proteins from humans and other mammalian sources; other insect viruses have also been studied as potential expression vectors. Examples of such pathogens include the entomopoxviruses, the bacterium Escherischia coli, and the fungus Pichia pastoris. Such pathogens may be enhanced as pest control agents by their modification to include toxin genes, much as the efficacy of baculoviruses has been enhanced by such modifications.
Thus it is clear that insecticidal toxins from arthropods may be used to advance the field of insect control in a number of significant ways. A novel composition of matter having the desired properties of insecticidal efficacy and insect selectivity, therefore, is expected to be useful in the art whether or not it can be used directly as an insecticidal compound. The means by which such a composition of matter may be made useful are well known to those skilled in the art, and are characterized by (but not limited to) the examples provided in the preceding paragraphs.
The venom of the wasp Bracon hebetor (also identified in the literature as Microbracon hebetor and Habrobracon hebetor) has been studied extensively due to its remarkable insecticidal potency. Thus, the present invention is directed to the isolation, purification, and identification of fractions of the venom of Bracon hebetor, and other species of the genus Bracon, which are useful in the study and control of insects.